Bioburden and Pyronema

ndustry Responds to Pyronema domesticum: HIMA Screening Studies Printer-friendly version Published: September 1, 1997 Find morecontent on: Industry Responds to Pyronema domesticum: HIMA Screening Studies Share Medical Device & Diagnostic Industry Magazine <a HREF="/mddi/by_issue.html">MDDI Article Index</a An MD&DI September 1997 Column Contamination of imported and domestic cotton products prompts investigation into effectiveness of EtO sterilization. During the summer of 1993, the Canadian Health Protectorate Branch found that some sterile laparotomy sponges from a U.S. manufacturer were contaminated with a fungal growth (or mold), later identified as Pyronema domesticum. The sponges, which had undergone EtO sterilization, were made from cotton that had been imported from China. Alerted to the situation, FDA and various manufacturers conducted tests of other lap sponges and China-origin cotton products, such as operating room towels, gauze, and surgical drapes, and found additional instances of Pyronema contamination. By early 1994, several voluntary product recalls had been initiated, and manufacturers of cotton products were investigating the organism and its resistance characteristics further.1,2 Although Pyronema is not recognized as a human pathogen and no infections were reported, this episode raised some serious concerns about current sterilization practices and potentially resistant microorganisms. As a result, on April 22, 1994, FDA's Center for Devices and Radiological Health (CDRH) distributed a memorandum addressed to "All Device Manufacturers/Repackers Using Cotton." Using terminology that included the words should and must, this document made a number of recommendations concerning sterilization validation for cotton medical devices. Upon receiving the memorandum, members of the sterilization and packaging subcommittee of the Health Industry Manufacturers Association (HIMA) decided to respond formally. Accordingly, a Pyronema Working Group was formed that included the authors of this article, and a written response was submitted to FDA on June 14, 1994. Despite undergoing sterilization, some cotton products have been contaminated by Pyronema domesticum. A total of 45 studies were performed by these manufacturers using 16 products (differentiated based on product description. with an average of 71. six manufacturers have submitted the results of their screening studies to HIMA for summary and analysis.4 Since that time.5 the screening procedure recommended that. the procedure was not intended as a replacement for bioburden testing. whenever possible. a study should be performed using various-sized sample portions to determine the minimum SIP needed to achieve bioburden recovery. The bioburden recovery validation distribution for the 16 products tested is shown in Table I.0. BIOBURDEN TESTING Based on accepted practices. . nor was it intended to replace a manufacturer's standard process validation protocol. All six manufacturers that submitted data followed the recommendation in the Pyronema screening procedure and tested whole products. In addition.4%. that is. the entire product should be used for bioburden testing rather than a sample item portion (SIP).3 Their reason for doing so was twofold: to provide a relatively simple protocol that manufacturers could use to detect the presence of Pyronema on their products. and radiation. one company shared the results of its validation studies on product inoculated with Pyronema domesticum. 1995.The working group also developed a draft screening procedure for Pyronema. For those manufacturers that reported validating their procedures. As stated in the "Scope" section of the draft. moist heat. The draft screening procedure further specified that the technique should be used for detecting naturally occurring Pyronema on product rather than organisms resulting from an artificial source. The draft screening procedure was sent to FDA for review and comment and then distributed in a letter format to HIMA members and nonmembers on February 1. which may not be representative of Pyronema's sterilization resistance in its natural state on products. while others did not provide this information. the SIP should equal 1. and product weight) and three sterilization modes: EtO. manufacturing plant. and to provide a consistent method that could be used to collect data to answer questions about the prevalence of Pyronema in cotton. bioburden recovery varied from 30 to 100%. The remainder of this article summarizes the study results and presents HIMA's recommendations regarding the sterilization of cotton medical products. such as an inoculum. The bioburden recovery procedures used by some manufacturers were reported as validated. When testing the entire product is not practical. and the one labeled 103 ranges from 500 to <5000). it is not known whether that recovery factor was used in the reported bioburden. The study results indicated that bioburden testing is of limited use in determining the presence of Pyronema. the numbers were added for total bioburden. All exposure temperatures were between 125° and 135°F with exposure times of 30 to 45 minutes for 28 of the studies and a 9-hour exposure for one . Unlike an immersion test performed following a partial sterilization cycle.) The data are shown in the figure in terms of bins. the bin labeled 102 ranges from 50 to <500. According to the cycle descriptions provided.) 3 19 Info. which represent various bioburden levels. Not Provided 4 25 Table I. Bioburden distribution data as submitted by the participating manufacturers.6:91. (Note: For those products in which a Pyronema recovery factor was reported. Each bin is labeled in terms of its mean value (1 X 10x). which is capable of isolating Pyronema.Bioburden Type Naturally occurring Inoculated % of total products Products Validated (No. was determined using the bioburden numbers as submitted. and the range on each bin is plus-or-minus half a log (for example. there was no general consensus on isolation of Pyronema using bioburden testing. For those companies that reported data separately for bacteria and molds. Bioburden recovery validation distribution for the 16 products tested.) 7 2 56 Products Not Validated (No. The six manufacturers performed a total of 29 studies in which 1950 units of 13 product types were sterilized using a sublethal EtO process. Figure 1. which is shown in Figure 1. ETO STERILIZATION TESTING Screening Studies. 100% EtO or 8.4% EtO: HCFC (hydrochlorofluorocarbon) mixtures were used in concentrations ranging from 535 to 800 mg/L. The bioburden distribution on products used for sterilization studies. The percentage of Pyronema positives was compared to the associated total bioburden count for each test group (in those cases where both pieces of information was provided).0 3–14 INPa 7–8 Table II. The source and processing of the raw materials as well as the procedures used to manufacture the products may have contributed to Pyronema remaining as part of the natural bioburden until sterilization processing. those data are shown in Figure 2.3 8. Information not provided. The results might have been caused by the test units having come from several manufacturers. however.study. but all reported data indicated that Pyronema domesticum survivors were detectable within 14 days. . that all 28°–32°C testing was P. No correlation should be drawn between high total counts and high levels of Pyronema positives. Results also suggest that more Pyronema survivors occur when sterilized products are incubated at temperatures between 20° and 25°C. Pyronema Positives/Total Units Tested Range % Pyronema Positive Average %Pyronema Positive Days to Detect Pyronema 20–25 28–32 20–25 a b 30–45 min. 30–45 min. Incubation times ranged from 14 to 30 days. of Studies No. Incubation Temperature (°C) Exposure Time No.0 22.5 0. All testing was performed using soybean-casein digest broth (SCDB) as the culture medium with incubation temperatures at two different ranges: 20°–25°C (25 studies) and 28–32°C (4 studies). Summary of EtO sterilization screening study data. A summary of the test data is shown in Table II. It should be noted. domesticum contamination rate had been as high as 78% in earlier EtO screening studies. 9 hrb 24 4 1 338/1500 1/350 8/100 0–98 0–1 8. Inoculated-Product Studies. These data reinforce the premise that Pyronema is more resistant to EtO than Bacillus subtilis BIs. However. showing Pyronema positives as a percentage of total counts. not Pyronema domesticum.2 to 37. and 72 product units were exposed for 5 minutes at 255°–260°F. which did not provide information on the rate of Pyronema detection. The units were incubated in SCDB for 14 days at 20°–25°C. Results of EtO screening studies. the reported data indicate that EtO may not be an effective mode of sterilization for product contaminated with resistant strains of Pyronema. All 10 of the product units tested produced Pyronema growth. One hundred product units that had been exposed for 5 minutes at 250°F were incubated in SCDB at 20°–25°C for 30 days. Bacillus subtilis biological indicators (BIs) and product inoculated with Pyronema domesticum (1100 ascospores and 10 sclerotia) were subjected to a 6-hour EtO sterilization cycle with a gas concentration of 450 mg/L. Of the 29 EtO sterilization screening studies. while all of the BIs were negative for growth. Inoculated-Product Study. The other manufacturer tested a product that had a 58% Pyronema domesticum contamination rate in EtO screening studies. Only two manufacturers provided screening results on sublethal moist-heat sterilization processing. These studies are the first documented evidence of Pyronema in cotton from a country other than China. Overall. Results also were submitted for two moist-heat studies in which products had been inoculated with Pyronema domesticum ascospores and sclerotia. caution should be used when attempting to relate the resistance of an organism as it naturally occurs to that of its lab-cultured counterpart. The organism in the contaminated domestic cotton cases was identified as Pyronema spp.. As in the screening studies using uninoculated product. Pyronema growth was detected within 7 days.0 minutes. Product units from the 10-minute cycle were split between SCDB and fluid thioglycollate medium and incubated at .performed by one manufacturer. In the validation study submitted by one manufacturer. 26 involved products made from cotton obtained from China and the remaining 3 were of products using cotton obtained from the United States. One manufacturer tested a total of 1200 units (representing four types of laparotomy sponges) that had been exposed to multiple sterilization cycles with F°s ranging from 21. Forty product units and standard moist-heat BIs (Bacillus stearothermophilus) were exposed for 10 minutes at 255°–260°F. No Pyronema growth was detected in either study. Figure 2. MOIST-HEAT STERILIZATION TESTING Screening Studies. 20°–25°C and 30°–35°C. 100 came from a batch of product that had demostrated a 44% P. respectively. Pyronema Positives/Total Units Tested 0/100a 0/150a % Positive 0 0 Days to Detect Pyronema N/Ab N/A Table III. Of these units. Summary of radiation sterilization screening study data from two manufacturers. b The third manufacturer performed studies in which two batches of 100 product units each were exposed to ever-increasing doses of radiation. All units were incubated in SCDB for 28 days. Three manufacturers submitted results from radiation processing screening studies. Pyronema domesticum was not detected in any of the product samples and all of the BIs were negative for growth. After 14 days of incubation. 250 product units were exposed to radiation doses ranging from 7. The exposure temperature ranged from 120° to 130°F with an exposure time of 45 minutes. Processed product units were incubated in SCDB at 20°–25°C for 14 days. Not applicable. Incubation Temperature (°C) 20–25 28–32 a No. and no Pyronema growth was detected. All product units from the 5-minute cycle were incubated in SCDB only. .6 to 12. Data from two of the manufacturers are summarized in Table III. In those two studies. RADIATION STERILIZATION TESTING Screening Studies. domesticum contamination rate in EtO screening studies. The EtO processing was performed with 100% EtO at a concentration of 600 mg/L. The results of these studies are shown in Table IV.8 kGy. followed by an EtO processing step to remove any organisms other than Pyronema that might have survived the radiation. 9 100/100 99/100 1/100 1/100 Table IV. The results of a 14. but no data were included as to the number or form (sclerotia or ascospore or both) of inocula used.e. Summary of data for screening studies that combined gamma radiation and EtO processing. Growth was detected within 7 days. however. it is difficult to relate the resistance of lab-cultured inocula to that of naturally occurring contamination by the same organism.6 N/A 4.4 0/100 N/A radiation sterilization study using inoculated a N/A= No radiation dose applied (i.1 9.3%) incubated in SCDB were positive for Pyronema. As mentioned previously.2 9. RECOMMENDATIONS .15 12.2 to 27.2 N/A 4.Gamma Radiation Dose (kGy) Batch 1 Batch 2 Pyronema Positives Total Units Tested 99/100 94/100 0/100 0/100 Minimum Maximum Mean Pyronema Positives Total Units Tested N/Aa 4. product units were submitted by one manufacturer..8 kGy.3 13.8 14. The product was inoculated with Pyronema domesticum. positive controls).0 14.0 12. After exposure to a radiation dose of 19. 14 out of 60 units (23.15 9. Inoculated-Product Studies. memorandum. bioburden should be assessed as part of the revalidation program. Periodically thereafter. 1994. Once this requirement has been met. Manufacturers intending to use EtO sterilization should perform a screening study to determine the presence (or absence) of Pyronema domesticum in their cotton products. All bioburden recovery methods should demonstrate the ability to recover a broad spectrum of microorganisms (bacteria.6 EtO sterilization may not be effective for cotton products containing Pyronema unless the cotton has been pretreated by a validated method effective against Pyronema domesticum. The 14-day incubation period currently used for immersion/sterility testing of cotton products is sufficient to detect Pyronema after exposure to a partial sterilization process. respectively. HIMA has issued recommendations covering the areas of bioburden testing. and routine sterilization processing. As stated above. placement of the microbial challenge should be in the hardest-tosterilize location within the chamber. including validation of the chosen SIP if whole-product testing cannot be performed. As with bioburden recovery methods. Sterilization of cotton products that have been shown in laboratory screening studies to be Pyronema free should be validated in accordance with ANSI/AAMI/ISO 11135. the chosen SIP must be validated. Products should be sterility tested as part of cycle development or validation.7.7. Sterilization of cotton products with radiation or moist heat should be validated in accordance with ANSI/AAMI/ISO 11137 and ANSI/AAMI/ISO 11134.8 CONCLUSION .6 All cotton products can be effectively terminally sterilized using a validated radiation or moist-heat process. yeast. the product can be effectively terminally sterilized using a validated EtO process.In response to the issues raised in FDA's April 22.5 For EtO and moist-heat cycle development and validation. Bioburden. sterility test methods should demonstrate the ability to recover a broad spectrum of microorganisms from the product. The following recommendations apply to all cotton products.8 Routine Sterilization. and molds) from the product. This pretreatment should be included as a part of the sterilization validation package (as part of the performance qualification). or if laboratory screening has shown the cotton product to be Pyronema free. and for testing using an SIP less than one. Sterilization Cycle Development/ Validation. sterilization cycle validation. The specific bioburden recovery method chosen for a given product should be validated.5 Bioburden should be assessed for products that are being used for sterilization cycle development studies. EtO sterilization may not be effective for cotton products containing Pyronema unless the cotton has been pretreated by a method effective against Pyronema domesticum. 8. Bryans T. Arlington. Association for the Advancement of Medical Instrumentation (AAMI). Manufacturers interested in obtaining a copy of the HIMA screening procedure can visit the association's Web site at or fax a request to Stacey Robertson at HIMA at 202/783-8750. 1991. 2. ANSI/AAMI/ISO 11134. Sterilization of Health Care Products—Requirements for Validation and Routine Control— Industrial Moist Heat Sterilization. Arlington." Med Dev Diag Indust. 16(10):102–109. 1994. AAMI. 6. DC. 1993. 1994. Washington.The working group concluded that cotton products containing Pyronema domesticum can be successfully sterilized if the process is validated in accordance with current ANSI/AAMI/ISO standards. VA. VA. ANSI/AAMI/ISO 11135. 8. VA. AAMI. "Industry's Struggle with Pyronema. ANSI/AAMI/ISO 11137. AAMI TIR no. Arlington. AAMI. Arlington. 5. and Aaronson J. 7. February 1. 4. "Screening Procedure for Pyronema" (draft). VA. Sterilization of Health Care Products—Requirements for Validation and Routine Control— Radiation Sterilization. Medical Devices—Validation and Routine Control of Ethylene Oxide Sterilization." Washington. 1994. 16(4):10. 16–20. Health Industry Manufacturers Association (HIMA). Joyce Hansen is affiliated with Sherwood/Davis and Geck. "Mold Impairs Sterile Cotton Industry." Med Dev Diag Indust. Microbiological Methods for Gamma Sterilization of Medical Devices. HIMA. 1995. 3. REFERENCES 1. 1994. Carol Lampe is affiliated with Baxter . Letter containing "Screening Procedure for Pyronema. Freiherr G. 1995. DC. . and Ann Baldwin is affiliated with HIMA.Healthcare Corp. Inc. a ViroMed biosafety company. Trabue Bryans is affiliated with Axios. Arnold Shechtman is affiliated with Medline Industries... Thelma Wilcott is affiliated with Becton Dickinson and Co.. Gerry O'Dell is affiliated with Johnson & Johnson Medical. Copyright ©1997 Medical Device & Diagnostic Industry . Inc.